The Human Genome Project (HGP) spurred a great increase in sequencing throughput and this, along with technical improvements, resulted in a corresponding drop in sequencing costs. In contrast to the 13 years and cost of nearly three billion US dollars, per genome sequencing costs have been reduced significantly—indeed two individual genomes have recently been completed (McGuire et al., Science 317:1687 (2007)). Personal genomes represent a paradigm shift in medical treatment for both patients and health care providers. By managing genetic risk factors for disease, health care providers can more readily practice preventative medicine and provide customized treatment. With large banks of completed genomes, drug design and administration can be more efficient, pushing forward the nascent field of pharmacogenomics.
Most conventional chemical or biochemical assays are based on “bulk” measurements. In such measurements, a collective behavior of a plurality of molecules within a certain volume of a sample solution is measured to determine the properties of the molecules. Recently, the detection of single molecule became possible. Single-molecule detection provides another option for chemical and biochemical assays, which offers much higher sensitivity and provides more detailed information than conventional bulk measurements, and soon became a new trend. An overview of the criteria for achieving single-molecule detection is discussed in, for example, the review articles by Moerner et al. (Moerner and Fromm, “REVIEW ARTICLE: Methods of single-molecule fluorescence spectroscopy and microscopy”, Review of Scientific Instruments 74(8): 3597-3619 (2003)) and Walter et al. (Walter, et al., “Do-it-yourself guide: how to use the modern single-molecule toolkit”, Nature Methods 5: 475-489 (2008)). These articles also discuss methods and apparatus that have been used or proposed for single-molecule detection.
U.S. Pat. No. 7,170,050 provides a zero-mode waveguide (ZMW) for single-molecule detection. The ZMW consists of a metal film and a plurality of holes formed therein, which constitute the core regions of the ZMW. In a ZMW, propagation of light having a wavelength longer than a cutoff wavelength in a core region is prohibited. When a light having a wavelength longer than the cutoff wavelength is incident to the entrance of the waveguide, the light will not propagate along the longitudinal direction of the core region. Instead, the light intensity will decay exponentially along the longitudinal direction of the core region, forming an evanescent field at the entrance of the waveguide. This offers a specific excitation zone, within which molecule is excited and the emitted fluorescent light is captured by confocal microscope. However, the detectable number of ZMWs is limited by the numerical aperture (NA) of the confocal microscope and the throughput is limited. U.S. Pat. No. 7,486,865 provides a recessed ZMW formed by extending the ZMW into the underlying substrate. This configuration allows a more tunable observation volume and higher signal level for optics placed below the waveguide. However, this configuration still has scale-up issue and limited throughput problems.
Therefore, there is a need for an apparatus to detect an object, especially an object emitting light of low intensity such as a single-molecule object.